Bioactive Compounds of Citrus Fruits: A Review of Composition and Health Benefits of Carotenoids, Flavonoids, Limonoids, and Terpenes

The increased consumption of fruits, vegetables, and whole grains contributes to the reduced risk of many diseases related to metabolic syndrome, including neurodegenerative diseases, cardiovascular disease (CVD), diabetes, and cancer. Citrus, the genus Citrus L., is one of the most important fruit crops, rich in carotenoids, flavonoids, terpenes, limonoids, and many other bioactive compounds of nutritional and nutraceutical value. Moreover, polymethoxylated flavones (PMFs), a unique class of bioactive flavonoids, abundantly occur in citrus fruits. In addition, citrus essential oil, rich in limonoids and terpenes, is an economically important product due to its potent antioxidant, antimicrobial, and flavoring properties. Mechanistic, observational, and intervention studies have demonstrated the health benefits of citrus bioactives in minimizing the risk of metabolic syndrome. This review provides a comprehensive view of the composition of carotenoids, flavonoids, terpenes, and limonoids of citrus fruits and their associated health benefits.


Introduction
Mechanistic, observational, and intervention studies have shown that increased consumption of fruits, vegetables, and whole grains contributes to the reduced risk of many diseases related to metabolic syndrome, including neurodegenerative diseases, cardiovascular disease (CVD), type 2 diabetes, and cancer [1,2]. These diseases are primarily associated with systemic and low-grade chronic inflammation prompted by oxidative stress. The bioactive compounds present in fruits, vegetables, and whole grains prevent the oxidative damage of cells by detoxifying the free radicals, thus minimizing the incidence of such diseases [3].
Citrus, the genus Citrus L. of the family Rutaceae, subfamily Aurantioideae [4], is one of the most important fruit crops, including pomelo, sweet orange, sour, lemon, lime, citron, grapefruit, kumquat, and hybrids [5,6]. The citrus fruit species widely investigated for their bioactive composition and their health benefits are listed in Table 1. Table 2. List of recently published outstanding reviews on composition and health benefits of citrus bioactive compounds.

Literature Search Methodology
Available electronic databases, especially Web of Science, PubMed, and Google Scholar, were searched for studies (review or experimental) that analyzed the composition of bioactive compounds in citrus fruits and their health benefits (in vitro, in vivo, and epidemiological). The primary search keywords were: (1) Citrus (title) and antioxidants (topic) or health (topic) and (2) Citrus (title) and bioactive (topic) or health (topic). The other keywords were: (1) Citrus (title) and flavonoid (title) or health (topic); (2) Citrus (title) and carotenoids (title) or health (topic); (3) Citrus (title) and essential oil (title) or health (topic); (4) Citrus (title) and essential oil (title) or health (topic); (5) Fruits (title) and health (topic); and (6) Diet (title) and health (topic). The relevant 320 articles were downloaded; they had been published mostly between 2018 and 2022. A total of 135 articles, including 128 published in the years 2022 (02), 2021 (37), 2020 (35), and 2019 (30), are discussed in this review.
PMFs are a unique class of bioactive flavonoids with more than two methoxyl (-OCH 3 ) groups on their chemical skeletons, and they abundantly occur in citrus fruits [29]. PMFs have attracted growing interest in recent years due to their anti-inflammatory [32], anti-atherosclerosis [33], anti-obesity [34,35], and anti-cancer properties [36]. Moreover, de-methylated PMFs, a product of fruit metabolism, chemical reactions during the drying process, and human metabolism, possess greater anticancer and anti-inflammatory activities than their corresponding methylated counterparts [37].
Deng et al. [38] isolated 11 flavonoids from (cv. Shatianyu) pulp; among them, naringin and rhoifolin showed the highest oxygen radical absorbance capacity (ORAC) activity. However, melitidin, bergamjuicin, and naringin were the major contributors to the ORAC activity in flavonoid extracts. In the albedo (inner layer) of ancient Mediterranean citrus fruit, flavonoids occupied 89.34% of polyphenolic fractions, dominated by flavanones eriocitrin and hesperidin as significant components, which accounted for 52.81% and 31.31% of the total flavonoids, respectively [30].
Citrus fruits contain the highest amount of flavonoids during the middle stages (60-80 days after pollination (DAP)) of development, and a decrease during complete maturation, probably due to the high expression of Chalcone synthase-1 (CHS-1) and chalcone isomerase, the rate-limiting enzymes in flavonoid biosynthesis [28,39,40]. In contrast, hesperidin peaked at the last developmental stage in the juice sacs of lemon (cv. Akragas) [41]. Moreover, the citrus fruit peel flavedo (outer layer) and the albedo contain more flavonoids than the juice sacs [41]. Among the 116 citrus accessions screened by Peng et al. [28], the highest amounts of PMFs, especially OCH 3 -PMFs (nobiletin and tangeritin), were recorded in loose-skin mandarins (including mandarins and tangerines) and their hybrids, followed by tangelo (C. reticulata × C. paradisi), sweet orange, junos, Rangpur lime, sour orange, and grapefruit [28]. Interestingly, the content of nobiletin, 5-demethylnobiletin, and tangeritin increased during the maturation and reached the highest at 60 DAP and decreased again . In the Persian lime, the highest amounts of flavanones (hes-peridin, 2005 µg/g; eriocitrin, 1171 µg/g; and narirutin, 1207 µg/g) and flavones (disomin, 366 µg/g; rhoifolin, 285 µg/g; and vitexin, 237 µg/g) were recorded at 12 weeks of growth and found to reduce at complete maturity (16 weeks). In contrast, in this study, the contents of flavanols (rutin and quercetin) were found to be highest at five weeks of maturation. In a comparative study, the highest amounts of total phenolic compounds were recorded in the albedo of unripe sweet orange (cv. Washington navel, 10910 mg kg −1 DW) and accounted for 50% of the cumulative content (flavedo + albedo + juice sacs), followed by orange (cv. Tarocco) flavedo, lemon (cv. Akragas) flavedo, and pummelo (cv. Chandler) albedo of unripe stages [41]. In this study, in the juice sacs of ripened fruits, flavanone hesperidin was the dominating phenolic compound in lemon (2213 mg/kg DW) and oranges (1957 and 1975 mg/kg DW in Washington novel and Tarocco, respectively), whereas flavanone narirutin was the most prevalent in pummelo (292 mg/kg DW). A significant amount of flavanone eriocitrin was recorded from lemon (913 mg/kg DW).
The presence of carotenoids and apocarotenoids confers the orange-red color to the peel and pulp of citrus fruits [47]. The carotenoid composition of citrus fruits is dominated by carotenoid fatty acid esters (xanthophyll esters) [44,45]. The occurrence of specific xanthophyll esters and total carotenoids largely depends on the species, maturity stage, and fruit parts [44,45]. For instance, at the fully mature stage, the total carotenoid contents of the flavedo of sweet orange were nine-fold higher (12.6 mg/100 g FW) than those in the pulp (1.4 mg/100 g FW) [45]. In this study, the most abundant carotenoids in the endocarp and flavedo of fully mature oranges were (all-E)-and (9Z)-violaxanthin, monoesters, and diesters esters carrying caprate, laurate, myristate, palmitate, stearate, palmitoleate, and oleate acyl moieties. The other major carotenoids were (all-E)-antheraxanthin, (all-E)-lutein, and (all-E)-β-carotene. In contrast, in this study, (all-E)-violaxanthin, (all-E)-lutein, (all-E)α-carotene, and (all-E)-β-carotene were also found to be prevalent in the flavedo of fully mature green fruits. Moreover, the esters of β-citraurin were also detected in the flavedo of fully mature oranges.
Similar to the phenolic compounds, the citrus fruit peel flavedo contains a higher amount of carotenoids than the juice sacs [41]. However, unlike phenolic compounds, carotenoid contents increase during maturation [41,50]. In addition, in contrast to phenolic compounds, the albedo contains only a trace amount of carotenoids [41]. In a comparative study among oranges (cv. Washington navel and cv. Tarocco), lemon (cv. Akragas), and pummelo (cv. Chandler), the highest amount of total carotenoids was recorded in the flavedo of ripened Washington navel orange (159 mg/kg DW), while, among the juice sacs of ripened fruits, the highest content of total carotenoids was recorded from Tarocco orange (63.7 mg/kg DW). In this study, lutein was the most dominating carotenoid in the juice sacs of the studied fruits, accounting for 83% of the total carotenoids in the juice sacs of Tarocco orange, whereas violaxanthin, antheraxanthin, β-cryptoxanthin, and β-carotene were minor carotenoids.

Essential Oil (Terpenes and Limonoids)
The essential oil obtained mainly from the flavedo of citrus fruits is an economically important product with beneficial health activities due to the presence of terpenes and limonoids with other bioactive components, including flavonoids, carotenoids, and coumarins [51,52]. The citrus essential oils are widely used in the pharmaceutical, cosmetics, perfumery, and food industries due to their natural fruity perfumes [11,53]. Moreover, citrus essential oils possess potent antioxidant, analgesic, anxiolytic, neuroprotective, and antimicrobial activities [11,54,55]. In particular, bioactive compounds from citrus essential oil are well known for their potential antimicrobial properties, as they cause significant lysis of the bacterial cell wall, intracellular ingredient leakage, and, subsequently, cell death [54]. Due to its potent antimicrobial activities, in recent years, the citrus essential oil has received significant attention as a preservation agent of fruits, vegetables, meat, and processed food products [53].
In the essential oil obtained from the fruit peel of Montenegrin mandarin, D-limonene and γ-terpinene were the major fractions, with the minor presence of citronellol and βlinalool [59]. Surprisingly, in this study, the presence of these minor components favored the antioxidant activity, while colorectal cancer HT-29 cells' cytotoxicity was significantly decreased. In a comparative study among essential oils obtained from grapefruit, lemon, mandarin, and orange, the highest 2,2 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical cation reducing activities and ferric reducing antioxidant power (FRAP) was obtained from mandarin essential oil, while lemon essential oil showed the highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging and cupric ion reducing antioxidant capacity (CUPRAC) [51].

Bioactive Compounds of Citrus Fruit Byproducts
The domestic and industrial processing of citrus fruit generates a considerable amount of peel, pulp, and seeds as byproducts, called pomace. A significant amount of research has been conducted to recover the commercially vital compounds from citrus fruit pomace [60][61][62]. Citrus peel is a rich source of essential oils [63], carotenoids [64][65][66], pectin [67,68], flavonoids [69][70][71], and several other bioactive components with excellent antioxidant [69] and health-promoting potential [62,[72][73][74]. Among the flavonoids, hesperidin, naringin, rutin, and neohesperidin are the major flavonoids found in the peel of citrus fruits [71,74], with especially high amounts in mandarins, which exhibit high antioxidant potency [71]. Surprisingly, the peel of most citrus fruits contains more polyphenols and other antioxidant compounds than edible pulp [65,75]. Therefore, peels from citrus fruits can potentially be used to recover these health-beneficial compounds. Moreover, given the low lignin content, the citrus peel can serve as a promising alternative to lignocellulosic biomass to produce biofuels [76].
Hesperidin and narirutin are the major flavonoids of C. unshiu peel [78,79]. Using the response surface model (RSM), the optimal extraction temperatures for the semi-continuous subcritical water extraction (SWE) of hesperidin and narirutin from C. unshiu peel were predicted as 164.4 • C and 154.6 • C, respectively, with an optimal flow rate of 2.25 mL/min. With these extraction conditions, the predicted yields of hesperidin and narirutin were 45.2 and 8.76 mg/g DW, respectively, corresponding to a recovery rate of 90.4% and 94.4%, respectively. In another study, Hwang et al. [78] optimized the extraction of hesperidin and narirutin from C. unshiu peel using SWE aided by pulsed electric field (PEF) treatments. In this study, PEF treatment for 2 min, combined with SWE at 150 • C for 15 min, provided the highest (46.96 mg/g DW) yield of hesperidin, while the narirutin yield was highest (8.76 mg/g DW) after PEF treatment for 2 min, combined with SWE at 190 • C for 5 min.
In view of the above, citrus pomace presents enormous opportunities to recover bioactive compounds and has a wide range of commercial applications in the food, feed, and pharmaceuticals industries. Moreover, the utilization of citrus pomace can create a surplus revenue that can substantially improve the economics of citrus fruit processing.
The pancreatic lipase (PL) is a crucial enzyme involved in triglycerides' hydrolysis in the gastrointestinal tract, and its inhibition can ameliorate obesity by minimizing lipid absorption [82]. Hesperidin, neohesperidin, naringin, narirutin, and eriocitrin were found to be the major components in the citrus peel extracts of grapefruit, pomelo, kumquat, mandarin, and ponkan [82]. Interestingly, in this study, among these flavonoids, hesperidin, the most dominant flavonoid in ponkan peel extract, showed the highest pancreatic lipase inhibition activities, suggesting its promising application in managing obesity.
Dysregulation of IL-5 secretion by antigen-specific T helper 2 (Th2) cells has been linked to eosinophilic inflammation in asthma [84]. The Th2 cytokine expression is regulated by transcription factors, including the nuclear factor of activated T cells (NFAT) [84].
Beyond the health-beneficial effects described above, bioactives present in citrus fruit juices were shown to have direct antiviral activity. Dong et al. [91] mentioned that hesperidin restricted the replication and progression of the Influenza virus in human lung carcinoma A549 cells by upregulating the p38 signaling pathway. Hesperidin, hesperetin, and naringenin were shown to inhibit key proteases involved in coronavirus replication and prevent virus entry into host cells [92][93][94].
The flavonoids (hesperidin, naringin, tangeritin, and rutin) rich in the hydro-ethanolic extract of C. reticulata Blanco peels have shown antiproliferative effects against BT-474 human breast carcinoma [95]. In this study, 500 µg/mL of extract treatment reduced the viability of BT-474 cells by 47% and 60% after 24 or 48 h of treatment, respectively.

Carotenoids
Carotenoids are widely investigated for their anticancer activities [96,97]. Carotenoids are well known for their antioxidant function in the normal cellular environment [42]. However, in cancer cells with an innately high intracellular ROS level, carotenoids may act as potent pro-oxidant molecules and promote ROS-mediated apoptosis [98]. In our study, we have demonstrated that the anticancer activities of β-cryptoxanthin derived from mandarin oranges on human cervical carcinoma (HeLa) cells are mediated through pro-oxidant action, which enhances the ROS generation, followed by the enhanced expression of caspase-3, -7, and -9, Bax, and p-53 at the mRNA, with the concordant suppression of antiapoptotic Bcl-2. These events trigger the nuclear condensation, loss of mitochondrial membrane potential, activation of caspase-3 proteins, and, finally, cleavage of nuclei DNA. In this study, β-cryptoxanthin substantially inhibited the proliferation of HeLa cells, with an IC50 value of 4.5 µM after 24 h of treatment.

Essential Oil (Terpenes and Limonoids)
The lumy essential oil rich in limonene (48.9%) and linalool (18.2%) has been shown to exhibit potent antioxidant and free radical scavenging properties with anti-acetylcholinesterase activities [86]. Moreover, in this study, lumy essential oil showed neuroactive effects by significantly reducing the burst frequency (MBR), assessed by the spontaneous electrical activity of rat cortical neuronal networks.
In the neuronal cells, K + imbalance, activation (phosphorylation) of extracellular signal-regulated protein kinase (ERK1), and reactive oxygen species (ROS) production are associated with the progression of Alzheimer's disease (AD) [87]. In addition, the acetylcholinesterase (AChE) enzyme involved in the hydrolysis of acetylcholine plays a vital role in triggering neuropsychiatric symptoms in AD [87]. Limonene has shown protective effects in Aβ 1-42 oligomer-triggered toxicity in primary cortical neurons (in vitro model of AD) by suppressing the AChE, ROS production, and voltage-gated K + channel KV3.4 hyperfunction, and downregulating phosphorylated (p)-ERK [87].
The coumarins isolated from pomelo have shown hepatoprotective activities in Dgalactosamine-treated normal human hepatic LO2 cells by suppressing the levels of alanine transaminase (ALT) and aspartate transaminase (AST), increasing the activities of antioxidant enzymes, including glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), and decreasing the level of malondialdehyde (MDA) [99].  ↓Bcl-2 mRNA, ↑Bax, caspase-3, -7, and -9 mRNA, nuclear condensation and disruption of the integrity of the mitochondrial membrane, activation of caspase-3 proteins, nuclei DNA damage, and apoptosis

In Vivo Studies
Excess caloric supply causes chronic hyperlipidemia and hyperglycemia, triggering atherosclerosis, hepatic steatosis, obesity, diabetes, and cardiovascular complications [102][103][104]. These metabolic diseases are linked to a wide array of metabolic complications [102]. Hyperlipidemia is the condition of disorder of lipid metabolism, resulting in abnormally elevated levels of low-density lipoprotein cholesterol (LDL-c) and very-low-density lipoprotein cholesterol (VLDL-c), triglyceride (TG), and total cholesterol (TC) in the blood, as well as reduced levels of high-density lipoprotein cholesterol (HDL-c) [102]. Similarly, chronic hyperglycemia is the condition of persistent and unusually high postprandial (after a meal) blood glucose levels, primarily due to the flawed insulin production [104]. Several recent animal studies have demonstrated the beneficial effects of citrus flavonoids, carotenoids, terpenes, limonoids, and other bioactives (e.g., pectin and coumarins) against metabolic syndrome (Table 4). Moreover, the potent antioxidant activities of citrus bioactives have shown protection against primary dysmenorrhea (PD) [105], pulmonary edema [80], cancer [36], and neuropsychiatric [106] and neurodegenerative diseases [107,108].

Flavonoids
The citrus flavonoids are most widely investigated for their antihyperglycemic and antihyperlipidemic effects in animal models [104,108]. Citrus flavonoids, such as hesperetin, have shown potential in attenuating hyperglycemia in streptozotocin (STZ)-induced diabetes in rats by releasing insulin from β cells of islets [104]. In this study, hesperetin supplementation of 40 mg/kg for 45 days showed a significant decrease in plasma glucose levels and a significant increase in the level of plasma insulin. It restored the compromised antioxidant status by increasing the activity of SOD, catalase (CAT), and glutathione peroxidase (GPx). Moreover, in this study, hesperetin alleviated hyperlipidemia by lowering the cholesterol, free fatty acid (FFA), TG, and phospholipid (PL) levels in diabetic rats, probably via the insulin-mediated reduction in the synthesis of fatty acids and cholesterol. Moreover, the authors suggested that the cholesterol-lowering effect of hesperetin is possibly due to the capability of hesperetin and other flavonoids to bind to bile acids, resulting in enhanced bile acid secretion and a reduction in cholesterol absorption [104].
The gastrointestinal microbiota composition plays a vital role in host physiology, nutrition, and metabolism [110]. Changes in the gastrointestinal microbiota composition, the community of pathogenic symbiotic and microorganisms, are probably responsible for the anti-obesity effects of citrus bioactives, especially flavonoids [35,111]. The abundance of gut microbiota, Firmicutes over Bacteroidetes, is linked to obesity-related metabolic syndrome [35]. Moreover, the gut microbiome's branched-chain amino acid (BCAA) metabolism is considered responsible for metabolic syndrome [35]. It is likely that microbially produced BCAAs, such as imidazole propionate, impair insulin signaling through the activation of mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and P70S6K [35]. Sterol regulatory element-binding proteins (SREBPs) play essential roles in regulating lipid homeostasis via mTOR. An extract rich in PMFs and hydroxy polymethoxyflavones (HOPMFs) (0.5% of HFD for 16 weeks) from citrus peel attenuated the obesity and modulated gut microbiota in male C57BL/6 mice fed a HFD by altering the gut microbiota, by increasing Prevotella and decreasing rc4-4 bacteria [111]. In this study, PMFs and HOPMFs alleviated the total body weight, decreased the lipids in 3T3-L1 preadipocytes, and reduced the adipocyte size and adipose tissue weight in the HFD mice. Moreover, in this study, PMFs and HOPMFs decreased the levels of lipid droplets (LD) and perilipin 1 protein and sterol regulatory element-binding protein 1 (SREBP-1) expression. Similarly, in another study, a citrus PMF (nobiletin and tangeretin)-rich extract was shown to ameliorate HFD-induced metabolic syndrome via gut dysbiosis (decreased Firmicutes-to-Bacteroidetes ratio), and regulated branched-chain amino acid (BCAA) metabolism [35]. In this study, the PMF-rich extract inhibited the phosphorylation of mTOR and P70S6K and decreased the expression of SREBPs in human liver HL-7702 cells and HFD-fed mice. Therefore, the authors hypothesized that the decreased BCAAs by the PMF-rich extract contribute to improving metabolic syndrome by inhibiting the mTOR/P70S6K/SREBP pathway.
Dietary administration of 0.05% PMF 5-demethylnobiletin has shown chemopreventive effects against azoxymethane/dextran sulfate sodium (DSS)-driven colorectal carcinogenesis in male CD-1 mice by reduced cell proliferation, increased apoptosis, and decreased mRNA and protein levels of proinflammatory cytokines IL-1β, IL-6, and TNF-α in the colon [36]. In this study, a significant amount of 5-Demethylnobiletin metabolites, namely 5,3 -didemethylnobiletin, 5,4 -didemethylnobiletin, and 5,3 ,4 -tridemethylnobiletin, was documented in the colonic mucosa of the treated mice. Surprisingly, these metabolites showed more potent effects than 5-demethylnobiletin on inhibiting the proliferation, inducing cell cycle arrest, and the apoptosis of HCT116 human colorectal cancer cells.
Higher levels of circulating thyroid-stimulating hormone (TSH) are vital for greater longevity [112]. The upregulation of sirtuin 1, which deacetylates transcription factors that contribute to cellular regulation, may positively upregulate the exocytosis of TSHcontaining granules [112]. Due to the antioxidant and anti-inflammatory properties, 15 mg/kg body mass (BM) of citrus naringenin has shown increased TSH secretion in 24-month-old male Wistar rats by upregulating the Sirt1 protein expression [112].
Hyperglycemia is considered a vital risk factor in developing neurodegenerative disorders, as it is known to promote brain astroglial activation, oxidative stress, inflammation, amyloid-β-accumulation, tau hyperphosphorylation, and memory impairment [108]. Tau hyperphosphorylation induces microtubule dysfunction, leading to the formation of neurofibrillary tangles (NFTs), which are often observed in AD [108]. The citrus auraptene and naringin have shown inhibitory effects against tau hyperphosphorylation, astroglial activation, and recovered the suppression of neurogenesis in the hippocampus of STZinduced hyperglycemic mice [108].
Chronic inflammation is involved in the etiology of several intestinal disorders, including inflammatory bowel diseases (IBDs), which mainly comprise ulcerative colitis and Crohn's disease [113]. The C. kawachiensis peel powder rich in flavonoids (naringin, narirutin, and auraptene) and dietary fiber protected from the DSS-induced intestinal inflammation in a murine model of colitis [113]. In this study, supplementation of peel powder (5% of diet, w/w) ameliorated the DSS-induced body weight loss, colon shortening, increased expression of pro-inflammatory cytokines (e.g., TNF-α), and decreased expression of colonic tight junctions (TJs) (e.g., occluding).

Carotenoids
The provitamin A carotenoids (e.g., β-cryptoxanthin) from citrus fruits have also shown effectiveness against metabolic syndromes, such as type 2 diabetes [103]. In the body (intestine and liver), provitamin A carotenoids are bio-converted to retinol by the activities of β-carotene 15,15 -oxygenase (BCO1). In a high-fructose-diet-induced type 2 diabetes model of Wistar male rats, feeding of citrus concentrate containing 0.086 mg βcryptoxanthin, 5.69 mg hesperidin, and 7.5 mg pectin for eight weeks decreased insulinemia, glycemia, and dyslipidemia by restoring the LDL-c and TG levels to be similar to the healthy group [103]. Moreover, in this study, feeding purified β-cryptoxanthin alone or with a matrix containing hesperidin and pectin showed the synergy between these constituents. Furthermore, in this study, β-cryptoxanthin from citrus fruits was shown to restore the vitamin A status in both control and prediabetic (high-fructose fed) rats; however, prediabetic rats showed lower absorption bioconversion of β-cryptoxanthin into retinoids.
The synergy between carotenoids and flavonoids is probably due to the enhanced uptake of carotenoids in the presence of flavonoid glycosides [114]. In Caco-2 cells, it has been shown that flavanone O-glycosylation (at C 7 of the A ring) led to the highest promoting effect on β-carotene absorption via enhanced paracellular permeability by transient drop-in tight junction (TJ) protein expression, and the upregulation of peroxisome proliferatoractivated receptor-gamma (PPARγ) and scavenger receptor class B type I (SR-BI; proteins involved in carotenoid absorption and transport) expression [114].

Essential Oil (Terpenes and Limonoids)
The overproduction of endometrial prostaglandins (PGs), especially prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2), is considered to be one of the critical factors for the progression of primary dysmenorrhea (PD) [105]. A higher ratio of PGF2α/PGE2 is considered to be a principal indicator of PD [105]. The citrus essential oil, particularly sweet orange essential oil rich in limonene, exhibited relief from estradiol benzoate-and oxytocin-induced PD in female Sprague Dawley rats via decreasing the level of PGF2α and increasing PGE2, resulting in a decrease in the ratio of PGF2α/PGE2. Moreover, in this study, citrus essential oil prevented a decrease in antioxidant status markers, including total antioxidant capacity (T-AOC), SOD, and CAT, and an increase in MDA levels.
Anxiety and depression are the most common forms of neuropsychiatric disorders [106]. The essential oil from oranges and its main component limonene have shown an antidepressantlike effect in a chronic unpredictable mild stress (CUMS) male Kunming mice mouse model by restoring the decreased curiosity and mobility, reduced body weight gain, reduced sucrose preference, decreased levels of monoamine neurotransmitter 5-hydroxytryptamine (5-HT), dopamine (DA), norepinephrine (NE), and brain-derived neurotrophic factor (BNDF) and its receptor tropomyosin receptor kinase B (TrkB) expression in the hippocampus, and increased levels of corticotropin-releasing factor (CRF) and corticosterone (CORT) [106].
The peel oil of mandarin, rich in limonene, myrcene, and carotenoids, has led to the dose-dependent growth inhibition of A549 non-small-cell lung cancer (NSCLC) cells and tumor growth in nude mice implemented with A479 cells [115]. In this study, supplementation of 5.25 mg/d of peel oil per mouse for seven days significantly decreased tumor growth by reducing the expression of membrane-bound Ras protein, increasing apoptosis, and inducing cell cycle arrest at the G0/G1 phase. ↓Plasma glucose, glycemia, insulinemia, and LDL-C, VLDL-C, and TG levels, ↑liver retinyl palmitate, and plasma β-cryptoxanthin [103]  ↓Bile acids in liver by increasing their efflux, ↓activation of HSCs by suppressing the expression of TGF-β1 and -SMAα and ↓expression of NF-κB, TNF-α, and IL-1β [116] Coumarin auraptene (5-20 mg/kg)  ↓Cell proliferation, ↑apoptosis, and ↓mRNA and protein levels of IL-1β, IL-6, and TNF-α in the colon [36]  ↓Adipocyte size, adipose tissue weight, and alleviated the total body weight, levels of lipid droplets, and perilipin 1 protein and SREBP-1 expression, ↑gut microbiota Prevotella, ↓rc4-4 bacteria [111] The

Human Studies
Similar to the in vitro and animal studies, case-control, cohort, and interventional studies have also demonstrated the health benefits of bioactive compounds derived from citrus fruits. A pooled meta-analysis of 14 case-control (13 hospital-based and two populationbased) and two cohort studies showed that people with the highest citrus fruit intake had a 50% reduction in risk of oral cavity and pharyngeal cancer compared to the lowest intake [123]. In this meta-analysis, the protective effect of citrus fruit was substantially higher in case-control studies (OR 0.47; 95% CI 0.40-0.55) compared to cohort studies (OR 0.73; 95% CI 0.55-0.96).
The oxidized (ox)-low-density lipoprotein (LDL) plays a vital role in converting macrophages to foam cells and the formation and progression of atherosclerotic lesions [124]. In a 3-month randomized, double-blind, controlled study, 23 untreated human subjects (16 males and seven females, mean age of 41.9 years) with cardiovascular risk (total cholesterol level >200 mg/dL and LDL-c > 130 mg/dl) consumed a commercially available flavonoid-rich hydroethanolic extract (Citrolive™; 1000 mg/day for 90 d) from bitter orange and olive leaf (Olea europaea L.), and they showed a significant reduction in ox-LDL-c and LDL-oxidase/LDL-c ratio and increased serum paraoxonase activity (PON1; athero-protective by preventing LDL oxidation) as compared to controls [124]. In another eight-week study of 96 healthy human subjects (51 intervention and 45 placeboes), supplementation of Citrolive™ (1000 mg/day for 8 weeks) improved endothelial function, as measured by flow-mediated vasodilation (FMD), reduced blood pressure and lipid metabolism-related parameters (TC, LDL-c, LDL-oxidase, oxidized/reduced glutathione (GSSH/GHS) ratio, protein carbonyl, and IL-6), and improved antioxidant and inflammatory status [125].
In a randomized, parallel, double-blind, placebo-controlled trial of 153 participants (53 women and 106 men; age 18 to 65) with pre-or stage-1 hypertensive conditions, supplementation of 500 mL/day of orange juice (containing 345 mg hesperidin) or hesperidinenriched orange juice (containing 600 mg of hesperidin) for 12 weeks reduced systolic BP (SBP; −6.35 and −7.36 mmHg) and pulse (PP) pressure. Interestingly, the SBP and PP decreased dose-dependently relative to the hesperidin intake.
Visvanathan and Williamson [126] reviewed acute (13 studies) as well as chromic (22 studies) human intervention studies of the effect of citrus fruits and juice intake on the risk of developing type 2 diabetes and concluded that the direct acute effect of citrus polyphenols on the postprandial glycemic response (a risk factor for type 2 diabetes) is subtle. However, citrus juice intake for longer periods (e.g., 500 mL/day for 12 weeks) showed improved fasting glucose, fasting insulin (9-32%), and insulin resistance.
The lower solubility hampers the bioavailability and microbial metabolism of flavonoids, thus probably yielding high inter-individual variability, resulting in inconsistent health benefits [127]. In a randomized crossover human pharmacokinetic study, 16 healthy subjects (eight men and eight women) were administered a single dose of 3.1 g lemon extract containing 260 mg eriocitrin (main flavanone of lemon) or 1.95 g orange extract containing 260 mg hesperidin (main flavanone of orange) and showed higher bioavailability of eriocitrin, compared to hesperidin, probably due to the higher solubility of eriocitrin [127]. Thus, the authors suggested that consumption of eriocitrin-rich lemon extract could provide better health benefits.
The emerging evidence has suggested that the bioactive compounds present in orange fruits are associated with the metabolism of the gut microbiota [128]. Brasili et al. [128] revealed that daily consumption of juice of cv. Cara Cara and cv. Bahia oranges, differing in vitamin C, flavanone, and carbohydrate content, affects the fecal microbiota and metabolome differently. Intake of Cara Cara orange juice increased the Mogibacteriaceae and Tissierellaceae families (Firmicutes), while the Odoribacteraceae family and the Odoribacter genus (Bacteroidetes) decreased. In contrast, in the Bahia group, the Enterococcaceae and Veillonellaceae families increased while the Mogibacteriaceae and Ruminococcaceae families and the Faecalibacterium prausnitzii decreased. The abundance of Mogibacteriaceae was found in healthy subjects.

Toxicity and Safety Profile of Citrus Fruit Bioactive Compounds
Bioactive compounds derived from citrus fruits have shown a good safety profile in animal toxicological evaluation. In a 90 d sub-chronic and acute oral toxicity study on Sprague Dawley rats, hesperidin isolated from the dehydrated peel of C. sinensis showed a low observed adverse effect level (LOAEL) at 1 g/kg, and a median lethal dose (LD 50 ) of 4.83 g/kg [130]. These observations suggest a good safety profile in the animals, as this concentration is much lower than the flavonoids administered in animal models (10-200 mg/kg) to obtain the health benefits (Table 4). Moreover, other citrus flavonoids, including nobiletin, tangeretin, and naringin, have shown a good safety profile [131,132].
The limonene, and other terpene-rich citrus flavor ingredients, such as oil, essential oil, whole fruit extract, and peel extract, are generally recognized as safe (GRAS) [133]. R-(+)-limonene has shown no observed adverse effect level (NOAEL) in rodents ranging from <75 to 500 mg/kg, and LD 50 values range from 4.40 to 6.60 g/kg [134].
Significant advancements have been made to study the composition, content, and health-promoting activities of citrus fruit bioactives. However, in future investigations, the following fields should be addressed to overcome the bottlenecks: (1) screening of traditional and new cultivars with modern analytical techniques to identify the genetic variation in content and composition; these data can help to select bioactive-rich cultivars for food formulations; moreover, precise identification of the bioactive-rich growth stage of citrus fruits suitable for consumption is necessary; (2) the elucidation of cellular and molecular mechanisms of functioning of citrus bioactives in the body; (3) more human interventional studies are required to demonstrate the health benefits of citrus bioactives; (4) the synergetic effects in the bioavailability and bioactivity among different citrus bioactive need more exploration; in addition, synergetic effects between citrus bioactives and clinically used drugs should be explored; (5) citrus fruit wastes can potentially serve as a low-cost and eco-friendly source of bioactives; however, further research is needed in the context of the efficient extraction and utilization of bioactives from citrus fruit waste.